1. Field of the Invention
The invention relates to a method and a device for disrupting a plant or animal sample for processing, i.e. for example the isolation of nucleic acids or proteins from the sample. Such preparations and tests are performed in a laboratory by a laboratory technician in accordance with standardized processing instructions. A so-called protocol is a part of such processing instructions. An example of such a protocol for isolating plasmid DNA from E. coli is apparent from document DE 101 53 957 A1.
2. Description of Related Art
In order to process a sample in the desired manner, i.e. isolate the nucleic acids or proteins, for example, so-called “kits” are commercially available depending on the sample and the desired result, such as the “UltraClean Tissue DNA Isolation Kit” by Qiagen (www.Qiagen.com). Prior to processing a sample using such a kit in accordance with a predefined protocol, the sample has to be prepared in a suitable manner.
Such typical preparations known from the prior art are being described below.
For example, an organ is removed from a laboratory animal, e.g. a rat. The selection of the organ of the animal depends on the objective. The removed organ or tissue of the animal is washed in a wash buffer solution such as in PBS (Phosphate Buffered Saline, with the following contents: Na2HPO4 (dried), NaH2PO4 (dried), NaCl and distilled water). Due to the washing process, the tissue of the removed part is provided in a blood-free state and is freed from undesired components.
Then, the removed tissue is cooled in liquid nitrogen, in order to stop cellular activities, among other things. Otherwise, the desired information would not be obtained in the desired quality subsequent to processing. Typically, tissue having a body temperature of, for example, 37° C. is submerged in liquid nitrogen in the process. Bubbles will develop. The tissue is withdrawn from the liquid nitrogen not until the formation of bubbles ceases. The tissue is then stored at −80° C. using, for example, dry ice.
If the cooling step in liquid nitrogen is to be avoided, then, as an alternative, the removed tissue is chemically preserved subsequent to the washing process, using stabilizing reagents such as RNAlater®, for example. RNAlater® is a viscous liquid which was developed by the company Ambion (www.ambion.com) for preserving fresh tissues. The preservative effect is primarily based on all enzymes being inactivated in the tissue by the removal of water, and on cellular activities being stopped. The viscous liquid has to diffuse quickly into all cells of the tissue. Therefore, the size of the pieces of tissues has to be limited to a side length of half a centimeter at most. Subsequent to the chemical treatment, the tissue thus treated is also cooled at −80° C. in order to store it thus until processing.
Typically, 10 to 100 mg of tissue is required for processing in order to be able to perform the desired test, isolation or the like. Prior to processing, the required amount of animal tissue is cut off using a scalpel, for example.
Individually or in combination, the sample preparation steps mentioned so far can be features of the invention described below.
The cut sample, that is, the cut tissue, is now disrupted, meaning that the cell walls have to be opened. This can be done mechanically, chemically or enzymatically. Mechanical disruption is carried out, for example, using a “TissueRuptor” by the company Qiagen, known from the TissueRuptor Handbook, July 2006, by Qiagen, Hilden Germany. In this case, a rotating blade disintegrates the cell walls of the tissue at 35,000 revolutions per minute. As a rule, mechanical disruptions are being carried out in a buffer in order to avoid damage to the ingredients, such as nucleic acids.
A mechanical disruption carried out in a container in the presence of a buffer, that is, a chemical substance, is known from document EP 1577011 A2.
Preparing samples in a cryogenic mill is also known (see for example http://www.laborpraxis.de/fachartikel/Ip_fachartikel_nh—2384859.html, Mar. 12, 2007). In this actively cooled mill, the sample is ground at the temperature of liquid nitrogen. The sample remains deep-frozen during the entire grinding process without coming into contact with the nitrogen. This technically quite complex method can be carried out in the case of such samples in which the above-mentioned method fails, for example in the case of very hard materials, such as bone, or of collagen-containing materials, such as skin. If a bone is to be prepared, putting it into a vessel filled with liquid nitrogen and crushing it using a metallic pin is also known. The bone is subsequently provided in a powdered form.
If histological tests with a microscope are to be carried out, then a sample is first impregnated with paraffin, hardened, and then cut into thin layers of tissue using a microtome.
If plant samples are to be processed, cutting them to size with a scalpel is only possible in the case of soft materials, such as leaves, soft beans etc. In the case of dried or frozen plant samples, they are frozen in liquid nitrogen and ground using a pestle in a mortar actively cooled with liquid nitrogen.
The German patent specification 738 286 teaches freezing and grinding cells together with a dispersion liquid in order thus to disintegrate the cells.
A crushing/mixing device for foodstuffs such as spices is apparent from each of the documents DE 602005001256 T2 and WO 2004/082837 A1. The device comprises a hollow body with a ball placed therein with which foodstuffs are to be crushed.
Documents US 2004/0144874 A1, JP 2006051505 A, JP 2002-066 366 A and JP 03-186 360 A disclose other examples for ball mills and comparable devices. No method for disrupting a biological sample is apparent from these documents.